The incidence of well-differentiated thyroid cancer has risen sharply over the past two decades and is expected to become the fourth most common cancer worldwide by 2030, with papillary thyroid carcinoma (PTC) being the most common type . When papillary carcinoma lacks typical lesions in histological structure and nuclear morphology, it is very difficult to differentiate it from benign lesions with similar morphology. For example, the follicular variant of papillary carcinoma (FVPTC) is macroscopically similar to encapsulated follicular adenoma, consisting of small- to medium-sized irregularly shaped follicles without papillary glia, and ground-glass-like nuclei may also be present in foci of follicular adenomas. Benign papillary hyperplasia lesions may have branched and complex papillary structures. The cells can appear vacuolated, the nuclei are enlarged and lightly stained, and occasionally, nuclear grooves are seen. Thus, it can be difficult to differentiate from PTC. The tumor cells in the remaining frozen specimens of PTC can be easily stretched and deformed, making histological diagnosis difficult. Papillary microcarcinoma (PTMC) may have only a few follicular lesions, thereby affecting clinical management. Traditional immune markers such as CK19, Galectin-3, and HBME-1 have low sensitivity or specificity for independent diagnosis, making microfocal lesions difficult to identify. In our daily work, we observed that CK19 was strongly expressed in PTC and was also positively expressed in normal thyroid tissue, but the positive intensity was slightly different. Therefore, some studies believe that the value of CK19 in the independent diagnosis of PTC is limited; it is only possible to suggest the existence of PTC when it is strongly positive . Although Galectin-3 has high sensitivity , it has poor specificity, as it is expressed in most benign thyroid nodules and chronic lymphocytic thyroiditis (CLT) . In the literature, the positivity rate of HBME-1 in PTC is more than 90% , but it also shows positivity in benign nodular thyroid lesions and benign papillary hyperplasia lesions; thus, it also has low specificity . Due to the diagnostic limitations of traditional immune markers, multi-index combined detection is often required to improve the diagnostic accuracy. O’Grady  found that when analyzing the high incidence of PTC, researchers focus only on the use of sensitive detection methods, resulting in overdiagnosis in some cases. At the same time, with the continuous discovery of new borderline thyroid lesions and benign and malignant “gray areas”, new immune markers with high sensitivity and specificity are urgently needed for auxiliary diagnosis.
The CCND1 gene was first identified in parathyroid adenomas due to the presence of a centromeric inversion of chromosome fourteen . This defect alters the structure of chromosomes, leading to uncontrolled cell proliferation. Cyclin D1 protein is overexpressed in various types of tumors . The most classic is the specific marker for mantle cell lymphoma (MCL) . In 2015, Lamba Saini  first proposed the use of Cyclin D1 as a diagnostic marker for “well-differentiated tumors of uncertain malignant potential (WDT-UMP)” and as a candidate marker for PTC. WDT-UMP is a lesion in follicular adenomatous nodules of the thyroid with focal nuclear features of PTC, which in this study is considered to be a precursor to FVPTC. Cyclin D1 is highly expressed in nuclear characteristic regions with focal PTC but not in surrounding adenomatous regions. However, the study only collected 13 groups of cases, so evidence from large data samples are still needed. Since the concept of WDT-UMP has not been widely accepted, borderline tumors—noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)—have been added to the WHO (2017) classification of endocrine tumors to define such lesions. However, Sora Jeon  found that the difference between NIFTP and FVPTC lies in the low expression of Cyclin D1b at the mRNA and protein levels. The function of Cyclin D1 in PTC is still unclear; more comprehensive histological sample studies are needed. Previous studies have found that P21 is closely related to tumorigenesis, and in vivo tumor model studies have revealed that P21-deficient mice exhibit sensitivity to hematopoietic, epithelial, and endothelial tumor formation . However, deletion of P21 in a mouse model of prostate cancer resulted in the reduced incidence and invasiveness of prostate cancer . The conflicting results of these studies suggest that P21 has dual roles in tumorigenesis and development, being either oncogenic or tumor suppressive depending on the tumor type and subcellular localization .
In our study, various types of histological samples were collected, and it was found that the expression of Cyclin D1 and P21 was localized in the nucleus. The positivity rate in PTC was much higher than that in adjacent tissues and other benign lesions (follicular adenoma and follicular epithelial papillary proliferative lesions), and the sensitivity (93%, 88.5%), specificity (96.06%, 99.28%), positive predictive value (94.41%, 98.89%), and negative predictive value (95.04%, 92.33%) were all high, which support their ability to accurately distinguish PTC from other similar benign lesions. The sensitivity of the combined detection of the two was 96.00%, which was significantly higher than that of the combined detection of the classical antibody markers CK19 + /HBME-1 + /Galectin-3 + (sensitivity 65.00%). The correlation analysis of clinicopathological characteristics showed that the expression of Cyclin D1 and P21 was positively correlated with tumor size and lymph node metastasis (P < 0.05), suggesting that Cyclin D1 and P21 may participate in the occurrence and development of PTC, the mechanism of which needs to be further explored. However, there was no correlation with sex, age, number of tumor foci, histological subtype, chronic lymphocytic thyroiditis or TNM stage, indicating that the expression of Cyclin D1 and P21 is not different in classical PTC and FVPTC. Differential expression of Cyclin D1 and P21 in PTC and follicular adenoma may help in differential diagnosis. Whether they can distinguish PTC in chronic lymphocytic thyroiditis from atypical nuclear characteristic lesions in chronic lymphocytic thyroiditis is one of the directions of our continuing research.
Lymph node metastasis is an important indicator of the prognosis of PTC. PTC metastases in cervical lymph nodes usually exhibit only a few follicular structures, and the identification of ectopic thyroid tissue is controversial. Studies have shown that thyroid tissue present in cervical lymph nodes is not always malignant . Therefore, the distinction between malignant and benign thyroid tissue in cervical lymph nodes is extremely important for treatment. Other investigators have proposed different morphological criteria for benign thyroid tissue, including benign thyroid tissue in lymph nodes that does not involve nodal parenchyma or multiple lymph nodes, along with the follicle size, a lack of papillary features of PTC, the absence of nuclear enlargement and crowded nuclei, and a lack of psammoma bodies . However, malignant transformation of ectopic thyroid tissue has also been reported . Therefore, PTC metastases in cervical lymph nodes from ectopic thyroid tissue can be easily misdiagnosed when only morphological diagnosis is used. Molecular testing of BRAFV600E, NRAS and KRAS and immunohistochemical markers HBME-1 and Galectin-3 has been helpful for the diagnosis of ectopic thyroid tissue, but these markers cannot clearly prove that the ectopic thyroid tissue is benign . Therefore, there is still a need to discover better markers to improve the diagnostic accuracy. Our study found that the sensitivities of Cyclin D1 and P21 for the identification of intralymph node PTC metastases and intralymph node ectopic thyroid tissue were 97.96% and 89.8%, respectively; the specificities were 73.33% and 86.67%, respectively; the PPVs were 92.31% and 95.65%, respectively; and the predicted values were 91.67% and 72.22%, respectively. The combined detection of Cyclin D1 + /P21 + had a sensitivity of 97.96% and a specificity of 73.33%. This indicates that Cyclin D1 and P21 have important value in the identification of intralymph node PTC metastases and intralymph node ectopic thyroid tissue.
The expression of Cyclin D1 is regulated by multiple factors. The Wnt/β-catenin signaling pathway drives the expression of the downstream key target gene CCND1 and was found to play an important role in the occurrence and development of hepatocellular carcinoma, being necessary for proliferation . Activation of Akt-dependent NF–κB/Cyclin D1 pathway promotes triple-negative breast cancer cell proliferation . GSK3β was involved in the development of cervical squamous cell carcinoma by negatively regulating the nuclear accumulation of Cyclin D1 , while p21 and p27 jointly stabilize the Cyclin D1 protein by inhibiting its nuclear export . The importance of Cyclin D1 in tumorigenesis and development and its regulatory mechanisms reflect the potential of CDK inhibitors in therapy. Since p21 has dual functions as a tumor suppressor and oncogene and is regulated by multiple pathways at different transcriptional and translational levels, the expression level of p21 should be strictly controlled , and its tumor suppressive or oncogenic function varies in different types of tumors. P21 regulates cell proliferation and metastasis by participating in the PI3K-Akt  and c-Myc-P21  signaling pathways. Consequently, the P21 protein expression level can affect the sensitivity of tumors to chemotherapy or radiotherapy. In head and neck squamous cell carcinoma, the overexpression of FXR1 binds and destroys P21 mRNA, destabilizing it and reducing p21 protein expression to promote tumor cell proliferation . In vivo studies have reported that silencing LincRNA  or MiRNA  leads to upregulation of p21 expression, thereby inhibiting tumor cell apoptosis and proliferation. The role of P21 in tumors is further complicated when P21 is used in combination with other CDK inhibitors. P16 and P21 were found to promote tumor growth by enhancing the chemotaxis of monocyte-derived suppressor cells (Mo-MDSCs) . Injection of the P21-P27 fusion protein into the MCF-7 cell line was reported to induce apoptosis and inhibit proliferation . These studies suggest that P21 plays an important role in tumor therapy. Regulation of the expression of P21 could be used as an adjuvant therapeutic approach for some specific types of tumors to inhibit tumor development or reduce drug resistance. Our study found that Cyclin D1 and P21 were both overexpressed in PTC. Spearman rank correlation analysis showed that there was a significant correlation between these two proteins, indicating that both must maintain proper nuclear accumulation and export and balance in their localization, activity and stability. To prevent and treat the occurrence of tumors, the underlying molecular mechanism should be clarified. At the same time, the results of this work are expected to provide new treatment ideas for clinicians treating patients with advanced thyroid cancer who are not sensitive to I131 treatment.
In conclusion, Cyclin D1 and P21 can accurately identify PTC and differentiate follicular adenoma from FVPTC. And it can assist in the diagnosis of intralymph node PTC metastases and intralymph node ectopic thyroid tissue. These markers could therefore serve as molecular targets for the clinical treatment of patients with advanced papillary thyroid cancer.